G protein-coupled receptor kinases (GRKs) are serine/threonine protein kinases best known for their ability to phosphorylate activated G protein-coupled receptors. Numerous studies have revealed that GRKs are important regulators of normal cell and organism function and contribute to a number of human diseases including cardiovascular disease, cancer and neurological disorders. While there has been significant progress in understanding GRK function, the mechanistic basis for many of the in vivo functions of GRKs remains largely unknown. During the last grant period, we initiated studies on the two C. elegans GRKs and found that they regulate a number of important biological processes including egg-laying, growth and sex determination. Interestingly, our work revealed that GRK-2 regulates serotonin metabolism and that GRK-1 and GRK-2 mediate opposing effects on egg-laying. Moreover, the serotonin metabolite 5-HIAA was found to bind to SER-1, a 5-HT2A receptor ortholog, and oppose the effects of serotonin. To further define the biological role of GRKs and provide mechanistic insight into how GRK structure correlates with in vivo function, we propose to use molecular, biochemical and cell biological strategies to better define the mechanistic basis for GRK regulation of egg-laying. Our central hypothesis is that these studies will enable us to mechanistically link GRK structure/function with important biological processes in the worm and that our findings will provide novel insight relevant to understanding the physiological and pathophysiological role of GRKs in humans. We plan to test our central hypothesis by pursuing three specific aims. In aim 1, we will elucidate the mechanisms involved in GRK-2 regulation of egg-laying and test the hypotheses that: (A) GRK-2 regulates serotonin metabolism by regulating vesicular monoamine transporter and/or monoamine oxidase activity and (B) 5-HIAA functions as a biased SER-1 agonist. In aim 2, we will elucidate the mechanisms involved in GRK-1 regulation of egg-laying and test the hypothesis that GRK-1 stimulates Go signaling. In aim 3, we will use molecular and proteomic strategies to define the in vivo interactions and substrates for C. elegans GRKs. We are poised to mechanistically dissect how GRK-2 regulates serotonin metabolism, how serotonin and 5-HIAA differentially modulate serotonin receptor function and how GRK-1 and GRK-2 coordinately regulate biology. These studies will provide important insight that is highly relevant to not only understanding GRKs in the worm but also to understanding how GRKs function in humans.